Tuesday, Apr. 11, 2000

Plan and Schedule for Examination and Conducting DNA Investigation of the Kennewick Remains

FPMcManamon

FINAL DRAFT-9 April 2000-FINAL DRAFT

The overall goal of this investigation is to extract, amplify, analyze, and interpret ancient DNA from the Kennewick remains to determine the mitochondrial DNA (mtDNA) haplogroups and Y-chromosome genetic characteristics of this individual as one component for assisting in the determination of cultural affiliation.

A. Examining the skeleton, determining condition of bone collagen or other suitable organic sources of DNA for sampling, and selecting a new sample (April-May 2000).

Following recommendations in Buikstra and Ubelaker (1994:95-106), indications of weathering, discoloration, polish, cut marks, rodent or carnivore gnawing, and other forms of modification of the skeletal elements will be observed, described, analyzed, and interpreted. Evidence of chemical and mechanical erosion due to surface exposure, extensive ground water, riverine exposure, and animal activity will be checked (Ubelaker 1989:96-107). The main objective will be to interpret postmortem treatment of the skeleton and subsequent cultural or natural environmental effects upon it. Bone surfaces will be inspected carefully for variation in color, staining, and other conditions. As appropriate and possible, rearticulation of bone elements will be done to aid in describing and analyzing these patterns of bone coloration and condition. Diagrams and scientific quality photos of these patterns will be completed and checked; color, staining, flaking, cracking, fracture patterns, etc., will be recorded using standard terminology and charts. Fracture patterns will be examined to distinguish old from more recent postmortem fracturing.

3. Micro-sampling for biochemical and physical analysis will be undertaken. This will involve the removal of small portions of bone for laboratory analysis to detect the level and condition of original organic material from which DNA could be extracted successfully. Probing, drilling, and some cutting of bone will be required. The damage to the remains will be kept to a minimum. There are at least two reasons for this. First, standard museum and scientific practice is to damage remains as little as possible to obtain necessary information. Second, handling or destruction of the remains is offensive to the Indian tribes with whom the Federal agencies have consulted on this matter.

The initial 14C measurement reported by the University of California, Riverside (UCR) laboratory in 1996 was undertaken on a sample of bone (5th left metacarpal) whose amino acid composition reflected a typical "collagen profile" and whose amino acid carbon content [AACC] (68.8% of that of a modern bone standard [MBS]) indicated a sample containing a significant amount of intact collagen (Taylor et al. 1998).

An interest in undertaking mitochondrial DNA (mtDNA) and Y-chromosome analyses has raised the question if Kennewick bone retains sufficient unaltered protein to perform successful DNA extractions. In light of the evidence for a significant range in protein preservation in the bones comprising the Kennewick skeleton, it is prudent to undertake appropriate biogeochemical and stable isotope analysis of microsamples from a wide range of bone to determine which bones might be prime candidates for DNA analyses.

The principal goal of the sampling is to obtain quantitative data to assist in selection of a new sample for mtDNA analysis.

Drs. Cassman, Larsen, Odegaard, Powell, Smith, Trimble, and Walker will consult with Dr. Taylor concerning the bone likely to be useful for the DNA sample. Dr. Taylor will concentrate his microsampling on these candidate bones. A preliminary estimate is that 10-20 microsamples may be needed to adequately assess the appropriate organic matter in the remains. However, the exact number of microsamples taken will be determined by the team considering physical anthropological, conservation, and curation concerns, as well as the requirement of the chemical testing. These samples will be transported to UC-Riverside for analysis.

The extent of racemization of aspartic acid, alanine, and leucine of each bone microsample will be obtained. This analysis follows upon the work reported by Poinar et al. (1996) which noted that in samples in which, for example, the D/Lasp value exceeds 0.08, ancient DNA sequences could be not retrieved. Poinar et al. (1999) more recently reported on the use of flash pyrolysis with GC/MS to identify certain pyrolysis products that can also be used as an index of the degree of DNA preservation.

The 13C and 15N measurements also will be obtained on the amino acid extracts from each microsample. These measurements will assist in the interpretation of the overall condition of the skeletal remains and the postmortem alterations they have experienced. These measurements also will provide more detailed and comprehensive data that will permit the resolution of the dietary signals reflected in the stable isotope values exhibited in these bones.

5. Dr. Smith and others have suggested special consideration be given to sampling of the organic material in the pulp cavity of the teeth of the Kennewick remains as a source of DNA. Powell and Rose (1999:5-6) describe the Kennewick teeth in detail. Most of the teeth are present, however, they are extremely worn. The CAT scans done of the teeth indicated that only two, the maxillary canines, have pulp chambers that are "not filled with either dentin or perhaps natural minerals from the burial environment." This information will be taken into account during the new examination of the remains and consideration of using the teeth for a DNA sample.

6. The analysis of the physical characteristics of the skeleton, completed during the 24-28 April examination and the chemical analysis done subsequently by Dr. Taylor and other experts at UC-Riverside will be used together to select a bone element, or a portion of an element from which a DNA sample can be removed. Dr. Taylor estimates that, if no unusual conditions are encountered, the chemical analysis should be completed within one week. However, if unexpected problems arise, the chemical analysis of the microsamples may take up to 30 days.

B. Identify and select laboratories to carry out DNA testing (April 2000)

1. NPS developed a list of questions (Decl. of Jason Roberts, Exhibit 2, Attachment 2) to ensure consistency and structure the discussions with potential DNA labs that we considered for this investigation. We used suggestions from experts who have given affidavits in the case, as well as the Bonnichsen plaintiffs, to create our list of DNA labs to contact. We contacted each of the DNA labs or experts listed below to determine their interest in analyzing a sample if we are able to obtain a good candidate bone. Our inquiries also confirmed the experience of each lab or expert in carrying out such research, and ascertaining their ability and willingness to conform to the tight time schedule we must follow to conclude the research in time for incorporating it into our analysis of cultural affiliation.

The labs and experts who we have contacted include:

Dr. Michael Brown, Emory University School of Medicine

Dr. Fredericka A. Kaestle, Yale University

Dr. D. Andrew Merriwether, University of Michigan

Dr. Dennis O'Rourke, University of Utah

Dr. Anne Stone, University of New Mexico

Dr. Sarah Tishkoff, The Pennsylvania State University

Drs. Noreen Tuross and Connie Kolman, Smithsonian

The most important factors in evaluating the information we have received from the laboratories are their experience with extracting, amplifying, and analyzing ancient DNA, their ability to undertake and complete the analysis, and their commitment to completing the necessary analysis within the time frame we have established in order to meet the court's deadline.

We have questioned each lab about the past analyses they have undertaken and how they ensure that contamination of different samples and amplifications is prevented. Of considerable importance has been their ability and willingness to complete the analysis by 1 August. We have chosen this date in order to provide ample time for the results of the DNA analysis to be incorporated with our other evidence concerning cultural affiliation.

Our initial discussions with Dr. Kaestle of Yale indicated a strong interest on her part and ability and willingness. We intend to discuss further with her a few additional issues. We have had interest expressed by Drs. Tuross and Kolman, who authored the review of what might be learned from DNA analysis of the Kennewick remains in this case. However, Dr. Tuross has been out of the country and we have been unable to complete our discussions with her. We believe that one of these two other labs will serve as our second independent lab for the analysis and will conclude our selection process within the next two weeks.

Of the others we have discussed this matter with, Drs. O'Rourke and Stone were interested by due to their schedules could not commit to completing the analysis within the tight time frame that we need to follow. Drs. Schurr and Tishkoff lacked immediate access to a lab set up for processing ancient DNA, and Dr. Brown's lab focused mainly upon analysis of modern DNA.

C. Conduct testing, analysis, and interpretation of the new samples at two independent labs (June- August 2000)

2. Following the selection of a bone or tooth for sampling new DNA samples, the sample will be hand carried to one of the laboratories where it will be carefully split into to halves. The second half then will be hand carried to the second lab.

3. The DNA labs will conduct extraction, ampflication, and analysis methods and techniques standard among experts investigating ancient DNA. Their efforts will focus upon mDNA and Y-chromosome DNA. Each selected lab will provide us with a description of their proposed methods, techniques, and analysis protocals. Their statement will address the concerns about ancient DNA analysis described in the report by Tuross and Kolman done as part of the Kennewick investigation (add ref)

D. Complete testing, analysis, and interpretation of the UC-Davis sample (May-August 2000).

2. The UC-Davis preliminary analyses in 1996 of one uncontaminated extraction from the metacarpal, together with additional preliminary studies, could minimize the amount of time needed to complete the UC-Davis analysis. In a preliminary proposal, Smith suggests using a month's time (possibly part of May and June) for initial evaluations of various samples of the bone. After this period, the completion of all possible DNA analyses might require as little as one additional month (e.g., in the event that contamination is not so severe a problem that sequencing of multiple clones to identify the source of such contamination is not necessary, that inhibitors do not appear that interfere with amplification are not co-extracted with the DNA, or that nuclear DNA cannot be successfully amplified for studies of Y chromosome marker ) or as long as five additional months (e.g. in the event that the above circumstances do not obtain).

References Cited

Poinar, H.N., Hess, M., J. L. Bada, and S. Paabo 1996. Amino acid racemization and the preservation of ancient DNA. Science 272: 864-866.

Poinar, H.N. and B. A. Stankiewicz 1999. Protein preservation and DNA retrieval from ancient tissues. Proceedings of the National Academy of Sciences 96:8429-8431.

Powell, Joseph and Jerry Rose 1999 Report on the Osteological Assessment of the "Kennewick Man" Skeleton (CENWW.97.Kennewick). Report on the Non-Destructive Examination, Description, and Analysis of the Human Remains from Columbia Park, Kennewick, Washington, edited by Francis P. McManamon, National Park Service, Department of the Interior, Washington, DC. [posted at www.cr.nps.gov/aad/kennewick; 14 October 1999].

Taylor, R. E., D. L. Kirner, J. R. Southon, and J. C. Chatters 1998. Radiocarbon Dates of Kennewick Man [Letter] Science 280:1171-1172.

Tuross, Noreen and Connie Kolman 2000 Potential For DNA Testing of the Human Remains From Columbia Park, Kennewick, Washington, National Park Service, Department of the Interior, Washington, DC. [posted at www.cr.nps.gov/aad/kennewick; 4 February 2000].

Ubelaker, Douglas H. 1989 Human Skeletal Remains: Excavation, Analysis, and Interpretation. Second edition, Taraxacum Publishing Company, Washington, DC.